Quercetin improves lacrimal gland function through its anti-oxidant actions: Evidence from animal studies, and a pilot study in healthy human volunteers.

Anti-oxidant properties of polyphenols have been gaining medical attention as a preventive factor against aging and/or lifestyle diseases. In this study, we examined the anti-oxidant activity of quercetin improved tear function through its effects on the lacrimal gland in mice and humans. Six week-old diabetic mice, a model for decreased tear production, were fed for 12 weeks ad libitum with an experimental diet containing 0.5% quercetin. As a result, the tear volume was significantly improved compared to the control, despite no changes in body weight, food intake, lacrimal gland morphology or biochemical serum parameters. Moreover, significantly higher SOD-1 and SOD-2 protein levels were detected in the lacrimal glands of quercetin-treated mice by western blot. In addition, quercetin treatment of mouse corneal cell lines exposed to oxidative stress resulted in dose-dependent inhibition of ROS production and enhanced cell survival. Finally, we examined quercetin pharmacokinetics, specifically its presence in serum and tears subsequent to onion consumption in healthy volunteers, and found that the distribution of quercetin and its metabolite shifted from serum to tear following onion intake. An improvement in tear film stability also resulted following the intake by these healthy volunteers of a new, quercetin-rich onion cultivar ("Quergold") in powder form. These results suggested that quercetin improved tear function through its effects on the lacrimal gland in mice and humans.

Identification of the Components in a Vaccinium oldhamii Extract Showing Inhibitory Activity against Influenza Virus Adsorption.

We previously reported that extracts from plants of the Ericaceae genus Vaccinium, commonly known as the kind of blueberry, inhibited the early steps of influenza virus (IFV) infection to host cells, and that the activity was correlated with the total polyphenol content. Particularly potent inhibitory activity was observed for Vaccinium oldhamii. In this study, we identified the active components in Vaccinium oldhamii involved in the inhibition of IFV infection. We sequentially fractionated the Vaccinium oldhamii extract using a synthetic adsorbent resin column. High inhibitory activity was observed for the fractions eluted with 30%, 40%, and 50% ethanol, and three peaks (peak A, B, and C) considered to represent polyphenols were identified in the fractions by HPLC analysis. Among these peaks, high inhibitory activity was detected for peak A and B, but not for peak C. These peaks were analyzed by LC/MS, which revealed that peak A contained procyanidin B2 and ferulic acid derivatives, whereas peak B contained two ferulic acid O-hexosides, and peak C contained quercetin-3-O-rhamnoside and quercetin-O-pentoside-O-rhamnoside. It is already known that these polyphenols have anti-IFV activity, but we speculate that ferulic acid derivatives are the major contributors to the inhibition of the early steps of IFV replication, such as either adsorption or entry, observed for Vaccinium oldhamii.

GH30 Glucuronoxylan-Specific Xylanase from Streptomyces turgidiscabies C56.

Endoxylanases are important enzymes in bioenergy research because they specifically hydrolyze xylan, the predominant polysaccharide in the hemicellulose fraction of lignocellulosic biomass. For effective biomass utilization, it is important to understand the mechanism of substrate recognition by these enzymes. Recent studies have shown that the substrate specificities of bacterial and fungal endoxylanases classified into glycoside hydrolase family 30 (GH30) were quite different. While the functional differences have been described, the mechanism of substrate recognition is still unknown. Therefore, a gene encoding a putative GH30 endoxylanase was cloned from Streptomyces turgidiscabies C56, and the recombinant enzyme was purified and characterized. GH30 glucuronoxylan-specific xylanase A of Streptomyces turgidiscabies (StXyn30A) showed hydrolytic activity with xylans containing both glucuronic acid and the more common 4-O-methyl-glucuronic acid side-chain substitutions but not on linear xylooligosaccharides, suggesting that this enzyme requires the recognition of glucuronic acid side chains for hydrolysis. The StXyn30A limit product structure was analyzed following a secondary ß-xylosidase treatment by thin-layer chromatography and mass spectrometry analysis. The hydrolysis products from both glucuronoxylan and 4-O-methylglucuronoxylan by StXyn30A have these main-chain substitutions on the second xylopyranosyl residue from the reducing end. Because previous structural studies of bacterial GH30 enzymes and molecular modeling of StXyn30A suggested that a conserved arginine residue (Arg296) interacts with the glucuronic acid side-chain carboxyl group, we focused on this residue, which is conserved at subsite -2 of bacterial but not fungal GH30 endoxylanases. To help gain an understanding of the mechanism of how StXyn30A recognizes glucuronic acid substitutions, Arg296 mutant enzymes were studied. The glucuronoxylan hydrolytic activities of Arg296 mutants were significantly reduced in comparison to those of the wild-type enzyme. Furthermore, limit products other than aldotriouronic acid were observed for these Arg296 mutants upon secondary ß-xylosidase treatment. These results indicate that a disruption of the highly conserved Arg296 interaction leads to a decrease of functional specificity in StXyn30A, as indicated by the detection of alternative hydrolysis products. Our studies allow a better understanding of the mechanism of glucuronoxylan recognition and enzyme specificity by bacterial GH30 endoxylanases and provide further definition of these unique enzymes for their potential application in industry.IMPORTANCE Hemicellulases are important enzymes that hydrolyze hemicellulosic polysaccharides to smaller sugars for eventual microbial assimilation and metabolism. These hemicellulases include endoxylanases that cleave the ß-1,4-xylose main chain of xylan, the predominant form of hemicellulose in lignocellulosic biomass. Endoxylanases play an important role in the utilization of plant biomass because in addition to their general utility in xylan degradation, they can also be used to create defined compositions of xylooligosaccharides. For this, it is important to understand the mechanism of substrate recognition. Recent studies have shown that the substrate specificities of bacterial and fungal endoxylanases that are classified into glycoside hydrolase family 30 (GH30) were distinct, but the difference in the mechanisms of substrate recognition is still unknown. We performed characterization and mutagenesis analyses of a new bacterial GH30 endoxylanase for comparison with previously reported fungal GH30 endoxylanases. Our study results in a better understanding of the mechanism of substrate specificity and recognition for bacterial GH30 endoxylanases. The experimental approach and resulting data support the conclusions and provide further definition of the structure and function of GH30 endoxylanases for their application in bioenergy research.

Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells.

The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation.

Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms.

Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm. sRAGE purified from the silkworm exhibited good stability and maintained specific ligand-binding ability. In addition, N-glycan analysis of sRAGE revealed that N-glucan completely lacked potentially allergenic fucose. Moreover, co-expression of biotin ligase (BirA) with C-terminal BioEase-tagged sRAGE in MSGs resulted in efficient biotinylation of sRAGE after addition of biotin bait. C-terminal biotinylated sRAGE could be immobilized onto a solid surface in one direction through binding to streptavidin without any loss of ability. The dissociation constant of sRAGE with fructose-BSA, a typical RAGE ligand, was 7.25 × 10-7 M, consistent with that on the mammalian cell surface. Thus, we developed a novel, innovative silkworm expression system for efficient expression of recombinant sRAGE, which could serve as a basis for the elucidation of RAGE-ligand interactions and facilitate the search for new ligands and inhibitors.

Isolation and structure determination of a new lantibiotic cinnamycin B from Actinomadura atramentaria based on genome mining.

New lantibiotic cinnamycin B was isolated from the extract of Actinomadura atramentaria NBRC 14695(T), based on genome mining and chemical investigation. The partial structure of cinnamycin B was established by 2D NMR experiments, which indicated that cinnamycin B had same methyl lanthionine bridging pattern with cinnamycin. The reduction with NaBH4-NiCl2 afforded the reduced cinnamycin B, and MS/MS experiment indicated the presence of hydroxy asparatic acid in the molecule. Cinnamycin B showed an antibacterial activity against Streptomyces antibioticus with dosage of 5 µg (0.5µL, 10 mg/mL solution) at spot-on-lawn testing method. The gene cluster of cinnamycin B on the genome of A. atramentaria was identified and discussed in comparison with that of cinnamycin.

Investigation of Supramolecular Nanofibers Formed from Multicomponent Nucleotide-Appended Bolaamphiphiles and Heteropolymeric DNA as a Template.

A novel DNA-templated multicomponent self-assembly of well-defined, molecular-level-controlled nanostructures in aqueous solution is demonstrated by using a thermal cycling procedure. The strategy uses four building blocks comprising 1,18-nucleotide-appended bolaamphiphiles (3'-phosphorylated adenosine, thymidine, guanosine, or cytidine connected to each end of an oligomethylene chain) and a heteropolymeric 58-mer containing a palindromic sequence as the template DNA. Atomic force microscopic observations, circular dichroism, and temperature-dependent absorption spectra revealed that the multicomponent self-assembly of the four nucleotide bolaamphiphiles and template DNA formed right-handed helical nanofibers with complementary base pairs during thermal cycling. Nanofibers were not formed if one of the four nucleotide bolaamphiphiles was missing, suggesting that construction of the helical nanofiber resulted from self-assembly of all four bolaamphiphiles to form matched base pairs sorted according to the sequence of the template DNA.

Estimated daily intake and seasonal food sources of quercetin in Japan.

Quercetin is a promising food component, which can prevent lifestyle related diseases. To understand the dietary intake of quercetin in the subjects of a population-based cohort study and in the Japanese population, we first determined the quercetin content in foods available in the market during June and July in or near a town in Hokkaido, Japan. Red leaf lettuce, asparagus, and onions contained high amounts of quercetin derivatives. We then estimated the daily quercetin intake by 570 residents aged 20-92 years old in the town using a food frequency questionnaire (FFQ). The average and median quercetin intakes were 16.2 and 15.5 mg day(-1), respectively. The quercetin intakes by men were lower than those by women; the quercetin intakes showed a low correlation with age in both men and women. The estimated quercetin intake was similar during summer and winter. Quercetin was mainly ingested from onions and green tea, both in summer and in winter. Vegetables, such as asparagus, green pepper, tomatoes, and red leaf lettuce, were good sources of quercetin in summer. Our results will help to elucidate the association between quercetin intake and risks of lifestyle-related diseases by further prospective cohort study and establish healthy dietary requirements with the consumption of more physiologically useful components from foods.

Isolation and structure determination of new siderophore albachelin from Amycolatopsis alba.

A new siderophore named albachelin was isolated from iron deficient culture of Amycolatopsis alba. The planar structure of albachelin was elucidated by the combination of ESI-MS/MS experiment and NMR spectroscopic analyses of the gallium (III) complex. The structure of albachelin was determined to be a linear peptide consisting of 6 mol of amino acids including 3 mol of serine, one mol each of N-α-acethyl-N-δ-hydroxy-N-δ-formylornithine, N-α-methyl-N-δ-hydroxyornithine, and cyclic N-hydroxyornithine. The stereochemistries of amino acids constituting albachelin were analyzed by applying modified Marfey method to the hydrolysate of albachelin. Based on bioinformatics, we deduced and discussed the possible biosynthetic gene cluster involved in albachelin biosynthesis from the genome sequence of A. alba. By prediction of substrates for adenylation domains, a non-ribosomal peptide biosynthetase gene (AMYAL_RS0130210) was proposed to be the main biosynthetic gene for albachelin biosynthesis. The related genes including transporter for siderophore were found near the NRPS gene as a gene cluster.

A NMR-based, non-targeted multistep metabolic profiling revealed L-rhamnitol as a metabolite that characterised apples from different geographic origins.

This study utilises (1)H NMR-based metabolic profiling to characterise apples of five cultivars grown either in Japan (Fuji, Orin, and Jonagold) or New Zealand (Fuji, Jazz, and Envy). Principal component analysis (PCA) showed a clear separation between the Fuji-Orin-Jonagold class and the Jazz-Envy class, primarily corresponding to the differences in sugar signals, such as sucrose, glucose, and fructose. Multistep PCA removed the influence of dominant sugars and highlighted minor metabolites such as aspartic acid, 2-methylmalate, and an unidentified compound. These minor metabolites separated the apples into two classes according to different geographical areas. Subsequent partial least squares discriminant analysis (PLS-DA) indicated the importance of the unidentified metabolite. This metabolite was isolated using charcoal chromatography, and was identified as L-rhamnitol by 2D NMR and LC/MS analyses. The remarkable contribution of L-rhamnitol to geographic discrimination suggests that apples may be characterised according to various factors, including storage duration, cultivation method, and climate.

Therapeutic effects of isoflavones on impaired salivary secretion.

Dry mouth, which is characterized by decreased salivation, has a number of causes; the involvement of estrogen has been suggested as symptoms typically develop in middle-aged females. However, there is a lack of consensus regarding the treatment of this condition. Soy isoflavones, a subgroup of flavonoids, are abundantly found in the soy germ. They are thought to exert a number of effects by specifically binding to estrogen receptors due to their structural similarity to estrogen. Recently, soy isoflavones have been found to exert antioxidant effects, ameliorating disorders caused by reactive oxygen/free radicals. Based on these observations, the effects of soybean isoflavones on impaired salivary secretion were studied in patients with dry mouth. Soy isoflavone aglycones were administered at 25 mg per day to 15 subjects with an average age of 67.9 ± 8.0 years for 2 months, and salivary secretion was analyzed. The results showed a significant improvement based on the saliva flow rate and self-completed questionnaire, thus suggesting the usefulness of isoflavones in improving the symptoms of salivary gland hypofunction.

Xylan-mediated aggregation of Lactobacillus brevis and its relationship with the surface properties and mucin-mediated aggregation of the bacteria.

Some Lactobacillus brevis strains were found to aggregate upon the addition of xylan after screening for lactic acid bacteria that interact with plant materials. The S-layer proteins of cell surface varied among the strains. The strains that displayed xylan-mediated aggregation retained its ability even after the removal of S-layer proteins. L. brevis had negative zeta potentials. A correlation between the strength of aggregation and zeta potential was not observed. However, partial removal of S-layer proteins resulted in decreases in the electric potential and aggregation ability of some strains. Therefore, xylan-mediated aggregation of L. brevis was considered to be caused by an electrostatic effect between the cells and xylan. L. brevis also aggregated in the presence of mucin, and the strengths of aggregation among the strains were similar to that induced by xylan. Thus, xylan- and mucin-mediated L. brevis aggregation was supposed to be caused by a similar mechanism.

Accumulation of supramolecular nanoparticles self-assembled from a bola-shaped cytidylic acid-appended fluorescein dye in cell nuclei.

We examined the cellular uptake of the nanoparticles self-assembled from a bola-shaped cytidylic acid-appended fluorescein derivative (C-FLU-C). The accumulation of fluorescence in the Caco-2 cell nucleus was observed mainly after the plateau phase of cell growth, indicating that C-FLU-C permeated the nuclear envelope without nuclear-localizing tags.

Construction of energy transfer pathways self-assembled from DNA-templated stacks of anthracene.

We describe optical properties of anthracene stacks formed from single-component self-assembly of thymidylic acid-appended anthracene 2,6-bis[5-(3'-thymidylic acid)pentyloxy] anthracene (TACT) and the binary self-assembly of TACT and complementary 20-meric oligoadenylic acid (TACT/dA20) in an aqueous buffer. UV-Vis and emission spectra for the single-component self-assembly of TACT and the binary self-assembly of TACT/dA20 were very consistent with stacked acene moieties in both self-assemblies. Interestingly, time-resolved fluorescence spectra from anthracene stacks exhibited very different features of the single-component and binary self-assemblies. In the single-component self-assembly of TACT, a dynamic Stokes shift (DSS) and relatively short fluorescence lifetime (τ=0.35ns) observed at around 450nm suggested that the anthracene moieties were flexible. Moreover, a broad emission at 530nm suggested the formation of an excited dimer (excimer). In the binary self-assembly of TACT/dA20, we detected a broad, red-shifted emission component at 534nm with a lifetime (τ=0.4ns) shorter than that observed in the TACT single-component self-assembly. Combining these results with the emission spectrum of the binary self-assembly of TACT/5'-HEX dA20, we concluded that the energy transfer pathway was constructed by columnar anthracene stacks formed from the DNA-templated self-assembly of TACT.

Tea polyphenols as novel and potent inhibitory substances against renin activity.

Renin inhibitory activities of three tea products were investigated for the first time in this work. Water extracts from fermented oolong and black tea showed strong renin inhibitory activities. By the means of ultrafiltration, gradient high performance liquid chromatography and spectroscopic analysis, four active compounds were separated from aqueous black tea extract and identified as theasinensin B, theasinensin C, strictinin, and a hexose sulfate with a galloyl moiety, which had IC50 values of 19.33, 40.21, 311.09, and 50.16 µM against renin activity, respectively. Further detection indicated that the potent inhibitor theasinensin B was present only in black tea, and that monomeric catechins did not contribute significantly to the renin inhibitory activities of tea products. These results revealed novel and potent tea-derived renin inhibitors and suggested another potential pathway for tea consumption to control hypertension.

Structure and biosynthesis of scabichelin, a novel tris-hydroxamate siderophore produced by the plant pathogen Streptomyces scabies 87.22.

Scabichelin and turgichelin, novel tris-hydroxamate siderophores, were isolated from Streptomyces antibioticus NBRC 13838/Streptomyces scabies JCM 7914 and Streptomyces turgidiscabies JCM 10429, respectively. The planar structures of scabichelin and turgichelin were elucidated by mass spectrometry, and 1- and 2-D NMR spectroscopic analyses of their gallium(III) complexes. The relative and absolute stereochemistry of the metabolites was determined by the modified Marfey's method in conjunction with computational modelling and NOESY NMR analysis of Ga-scabichelin and Ga-turgichelin. Genome sequence analysis of the plant pathogen Streptomyces scabies 87.22 identified a gene cluster containing a gene encoding a nonribosomal peptide synthetase (NRPS) that was predicted to direct the production of a pentapeptide with structural similarities to scabichelin and turgichelin. Comparative LC-MS/MS analyses of iron-deficient culture supernatants from wild type S. scabies 87.22 and a mutant in which the NRPS gene had been disrupted, and scabichelin purified from S. antibioticus, showed that scabichelin is the metabolic product of the cryptic gene cluster, strongly suggesting that it functions as a siderophore.

Comparative analysis of flagellin glycans among pathovars of phytopathogenic Pseudomonas syringae.

Flagellin is a principal component of the flagellum filament. Previously, we reported that the flagellin of Pseudomonas syringae pv. tabaci 6605 (Pta6605) was glycosylated by oligosaccharides composed of two or three l-rhamnosyl (l-Rha) residues and a terminal 4,6-dideoxy-4-(3-hydroxybutanamide)-2-O-methylglucopyranosyl residue. In this study, we characterized the chemical structure of flagellin glycans in P. syringae pathovars glycinea race 4 (Pgl4), phaseolicola 1448A (Pph1448A), tomato DC3000 (PtoDC3000), and syringae B728a (PsyB728a). Flagellin glycans were released by hydrazinolysis, labeled on the reducing ends with 2-aminopyridine (PA), and the PA-labeled oligosaccharides were isolated by high-performance liquid chromatography. The purified PA-labeled glycans were analyzed by mass spectrometry and NMR spectroscopy. The results showed that the glycans on flagellin of Pgl4, PtoDC3000, and Pph1448A were identical to those of Pta6605, which were characterized previously. The flagellin of PsyB728a is O-glycosylated with a novel trisaccharide identified as 2-acetamide-2-deoxy-ß-D-glucopyranosyl-(1→2)-3-O-methyl-α-L-rhamnopyranosyl-(1→2)-L-rhamnose. Our data indicate that flagellin glycosylation of P. syringae pathovars has universality with little diversity.

Defects in D-rhamnosyl residue biosynthetic genes affect lipopolysaccharide structure, motility, and cell-surface hydrophobicity in Pseudomonas syringae pathovar glycinea race 4.

D-rhamnose (D-Rha) residue is a major component of lipopolysaccharides (LPSs) in strains of the phytopathogen Pseudomonas syringae pathovar glycinea. To investigate the effects of a deficiency in GDP-D-rhamnose biosynthetic genes on LPS structure and pathogenicity, we generated three mutants defective in D-Rha biosynthetic genes, encoding proteins GDP-D-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose reductase (RMD), and a putative α-D-rhamnosyltransferase (WbpZ) in P. syringae pv. glycinea race 4. The Δgmd, Δrmd, and ΔwbpZ mutants had a reduced O-antigen polysaccharide consisting of D-Rha residues as compared with the wild type (WT). The swarming motility of the Δgmd, Δrmd, and ΔwbpZ mutant strains decreased and hydrophobicity and adhesion ability increased as compared with WT. Although the mutants had truncated O-antigen polysaccharides, and altered surface properties, they showed virulence to soybean, as WT did.

Effects of lipooligosaccharide inner core truncation on bile resistance and chick colonization by Campylobacter jejuni.

Campylobacter jejuni is the most common bacterium that causes diarrhea worldwide, and chickens are considered the main reservoir of this pathogen. This study investigated the effects of serial truncation of lipooligosaccharide (LOS), a major component of the outer membrane of C. jejuni, on its bile resistance and intestinal colonization ability in chickens. Genes encoding manno-heptose synthetases or glycosyltransferases were inactivated to generate isogenic mutants. Serial truncation of the LOS core oligosaccharide caused a stepwise increase in susceptibilities of two C. jejuni strains, NCTC 11168 and 81-176, to bile acids. Inactivation of hldE, hldD, or waaC caused severe truncation of the core oligosaccharide, which greatly increased the susceptibility to bile acids. Both wild-type strains grew normally in chicken intestinal extracts, whereas the mutants with severe oligosaccharide truncation were not detected 12 h after inoculation. These mutants attained viable bacterial counts in the bile acid-free extracts 24 h after inoculation. The wild-type strain 11-164 was present in the cecal contents at >10(7) CFU/g on 5 days after challenge infection and after this time period, whereas its hldD mutant was present at <10(3) CFU/g throughout the experimental period. Trans-complementation of the hldD mutant with the wild-type hldD allele completely restored the in vivo colonization level to that of the wild-type strain. Mutants with a shorter LOS had higher hydrophobicities. Thus, the length of the LOS core oligosaccharide affected the surface hydrophobicity and bile resistance of C. jejuni as well as its ability to colonize chicken intestines.